Sigma ligands for neuronal regeneration and functional recovery

ABSTRACT

The invention discloses methods and compositions useful for facilitating neuronal regeneration and functional recovery in neurodegenerative diseases. The methods and compositions utilize ligands for the sigma receptor, wherein the ligand is preferably siramesine, or salts, or solvates thereof. These molecules can be delivered alone or in combination with agents which treat or prevent neurodegenerative diseases such as those caused by ischemic stroke, diabetic peripheral neuropathy, cancer therapy induced neuropathy, multiple sclerosis, amyotrophic lateral sclerosis, traumatic brain injury, spinal cord injury, Huntington&#39;s disease or Parkinson&#39;s disease. In other methods, the sigma receptor ligands are administered after stroke to facilitate functional recovery. The administration of the sigma receptor ligands effects faster functional recovery.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional patentapplication No. 60/478,735 filed on Jun. 12, 2003, No. 60/478,329 filedon Jun. 12, 2003, No. 60/498,132 filed on Aug. 26, 2003 and No.60/552,613 filed on Mar. 12, 2004, all of which are herein incorporatedby reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to methods of treatment to achieveneuronal regeneration in subjects with neurodegenerative disorders. Inparticular, the present invention relates to the use of sigma receptorligands to facilitate neuronal regeneration and functional recovery insubjects after neurodegenerative disease.

BACKGROUND OF THE INVENTION

The existence of the sigma receptor was proposed by Martin et al. (1976)J. Pharmacol. Exp. Ther. 197: 517-532 to explain the psychotomimeticeffects of benzomorphans. Initially, the sigma receptor was thought tobe a novel opioid receptor. However, the binding of the benzomorphans tothe sigma receptor is not antagonized by naloxone, the classic opioidreceptor antagonist. Further, the benzomorphans bind to a site that isdistinct from the phencyclidine receptor on the N-methyl-D-aspartate(NMDA) receptor complex. Thus, the sigma receptor is established as aunique receptor.

The sigma receptor consists of two subtypes, named sigma-1 and sigma-2.Hellewell and Bowen (1990) Brain Res., 527: 224-253 were the first todefine the characteristics of the two putative sigma receptor subtypes.The primary pharmacological distinction between these two sites is theaffinity of the (+) isomers of the benzomorphan opiates for the bindingsites. These compounds, such as (+)SKF 10,047 (NANM) and (+)pentazocineshow nearly two orders of magnitude higher affinity for the sigma-1 sitecompared to the sigma-2 site. The (−) isomers of the benzomorphans showlittle selectivity between these two sites. Other distinctions notedbetween the two sites are a preponderance of the sigma-2 sites in celllines such as NCB-20, PC12 and NG108-15 cells (Hellewell and Bowen;Quirion, R., et al., (1992) Trends in Pharmacological Sciences, 13:85-86). The sigma-1 receptor has been identified and cloned, but not thesigma-2 receptor (Langa et. al., European Journal of Neuroscience, Vol.18., 2003, pages 2188-2196). The endogenous ligands for sigma receptorsare unknown.

The subcellular distribution of sigma-1 receptors in brain includes thehippocampus, cortex layer and olfactory bulb. Sigma-1 is a 26 kDaprotein, and the gene encoding the receptor has been cloned. Hydropathyanalysis suggested that the sigma-1 receptor has two transmembranesegments. Further, the sigma-1 receptors share no homology with anyother known mammalian proteins.

Both types of the sigma receptors are expressed in the central nervoussystem as well as in peripheral tissues. Therefore, ligands for thereceptor could be used for the treatment and prevention ofneurodegenerative diseases. Consequently, brain sigma receptors havebeen the subject of intense investigation (Sonders et al. (1988) TrendsNeurosci., 1: 37-40). In general, sigma receptors exhibit promiscuousbinding to a wide variety of ligands such as psychotic drugs,antidepressants and neurosteroids. They have been demonstrated to playimportant roles in learning and memory in animal models of amnesia aswell as in behavioral models of depression. Numerous studies havedemonstrated robust neuroprotective properties of sigma receptor ligandsin animal models of cerebral ischemia. The mechanism of neuroprotectionfor some of these sigma ligands has been controversial because both thesigma receptors and the phencyclidine (PCP) binding sites of the NMDAreceptor channel complex have been reported to contribute to theseeffects.

Neurodegenerative diseases are characterized by the dysfunction anddeath of neurons, leading to the loss of functions mediated by thebrain, spinal cord and the peripheral nervous system. These disordershave a major impact on society. For example, approximately 4 to 5million Americans are afflicted with the chronic neurodegenerativedisease known as Alzheimer's disease. Other examples of chronicneurodegenerative diseases include diabetic peripheral neuropathy,multiple sclerosis, amyotrophic lateral sclerosis, traumatic braininjury, spinal cord injury, Huntington's disease and Parkinson'sdisease. Normal brain aging is also associated with loss of normalneuronal function and may entail the depletion of certain neurons.

Stroke is the third ranking cause of death in the United States, andaccounts for half of neurology inpatients. Depending on the area of thebrain that is damaged, a stroke can cause coma, paralysis, speechproblems and dementia. The major causes of cerebral infarction arevascular thrombosis, cerebral embolism, hypotension, hypertensivehemorrhage, and anoxia/hypoxia. However, the adult brain retainscapacity for plasticity and functional reorganization throughout thelife span, even after stroke or brain ischemia. Neuronal connections arecontinuously remodeled. The potential capability of the brain tocompensate for the damaged part of the brain has relevance for strokerehabilitation. Neuroimaging in stroke patients suggests some functionalreorganization. Thus, one aspect of brain plasticity is that in strokepatients, the neuronal connections can be modified by sensory input,experience and learning, and the brain can respond by functional andstructural reorganization, upregulation or downregulation of a neuralresponse to an event, and the establishment of new functional andstructural connections by collateral sprouting and compensatorysynaptogenesis, as well as neurogenesis.

However, aside from the effect of the environmental factors on brainplasticity, drugs and the interactions between drugs and environmentalfactors are another aspect to be considered. Thus, the need continues toexist for new drugs and new methods for the treatment of central nervoussystem disorders and other conditions that take advantage of brainplasticity to assist neuronal regeneration and functional recovery. Thepresent invention fulfills these and other needs.

Several sigma receptor ligands have been found to be neuroprotective(i.e. to protect against neuronal cell death and consequential loss offunction) in predictive models used for the testing of drugs forneuroprotective activity. For example, the sigma receptor ligandopipramol was found to protect against ischemia in gerbils and was foundto modulate the NMDA-type of glutamate receptors. In addition, othersigma ligands, including BMY-14802, caramiphen and haloperidol,exhibited properties in in vivo models that were consistent withaffording protective effects against NMDA-induced toxicity and seizures(M. Pontecorvo et al., (1991) Brain Res. Bull., 26:461-465), and severalsigma ligands were found to inhibit ischemia-induced glutamate releasefrom hippocampal slice preparations in vitro (D. Lobner et al., (1990)Neuroscience Lett., 117:169-174).

U.S. Pat. No. 5,736,546 discloses certain 1,4-(diphenylalkyl) piperazinederivatives that are ligands for sigma receptors. One of the compounds,1-(3,4-dimethoxyphenethyl)-4-(3-phenylpropyl) piperazine, is now alsoknown as SA4503. Nakazawa et al., Neurochem. Int., 32 (1998), 337-343report that SA4503 is a selective sigma-1 agonist and was found tosignificantly suppress hypoxia/hypoglycemia-induced neurotoxicity in ratprimary neuronal cultures. This neuroprotective action led the authorsto suggest that sigma-1 receptors may be useful in the treatment ofneurodegeneration (see page 342). Senda et al., European Journal ofPharmacology, 342 (1998), 105-111 further report that SA4503 was foundto be active against glutamate neurotoxicity in cultured rat retinalneurons. The authors suggest that sigma-i receptor agonists may beuseful against retinal diseases with neuronal cell death due toischemia, such as central and branch retinal artery occlusion, diabetesmellitus, age-related macular degeneration, hemoglobinopathies andvarious types of glaucoma. SA4503 is currently undergoing clinicaldevelopment for the treatment of depression, and has also been noted ashaving potential use in the treatment of dementia and drug dependence.

U.S. Pat. No. 5,665,725 discloses certain piperidine derivatives thatare ligands for sigma receptors. The compounds are said to be useful inthe treatment of anxiety, psychosis, epilepsy, convulsion, movementdisorders, motor disturbances, amnesia, cerebrovascular diseases, seniledementia of the Alzheimer type and Parkinson's disease. One of thecompounds, 1′-[4-[1-(4-fluorophenyl)-1H-indol-3-yl]-1-butyl] spiro[isobenzofuran-1(3H),4′-piperidine], is also known as Lu 28-179 orsiramesine. It is a selective sigma-2 agonist and also displays activitytowards the sigma 1 receptor (Perregaard J., et al., J. Med. Chem.,1995, 38, pages 1998-2008). International patent application,publication number WO 99/24436 further discloses that the hydrohalidesalts of the compound, in particular the hydrochloride salt, have goodbioavailability.

Thus the art suggests that sigma ligands may be useful asneuroprotective agents in the treatment of subjects withneurodegenerative diseases.

Unexpectedly, it has now been found that certain sigma ligandsfacilitate functional recovery in subjects suffering fromneurodegenerative disease. Thus, the sigma ligands are useful asneuroregenerative agents in the treatment of neurodegenerative diseasefollowing a neuronal insult.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions for treatingneurodegenerative diseases. The sigma receptor ligands of the inventionenhance functional recovery and neuronal regeneration. These moleculescan be delivered alone or in combination with additional agents, and areused as neuronal regeneration agents for the treatment ofneurodegenerative diseases such as those resulting from ischemic strokesor other insults that injure neurons.

Accordingly, in one aspect, the subject invention is directed to amethod for treating or preventing neurodegenerative disease in a subjectin need thereof. The method comprises administering to the subject apharmaceutically effective amount of a ligand for the sigma receptor.

The invention thus provides methods for treating neurodegenerativedisease in a mammalian subject in need thereof to facilitate neuronalregeneration leading to functional recovery after a neurodegenerativedisease, the method comprising administering a pharmaceuticallyeffective amount of a sigma receptor ligand to the subject.

In another aspect, the present invention provides the use of a sigmaligand in the manufacture of a medicament to facilitate neuronalregeneration leading to functional recovery in a mammalian subject aftera neurodegenerative disease.

In yet another aspect, the present invention provides a pharmaceuticalcomposition, which comprises a sigma ligand for treating a mammaliansubject to facilitate neuronal regeneration leading to functionalrecovery after a neurodegenerative disease.

The neurodegenerative disease can be ischemic stroke, Alzheimer'sdisease, diabetic peripheral neuropathy, cancer therapy inducedneuropathy, multiple sclerosis, amyotrophic lateral sclerosis, traumaticbrain injury, spinal cord injury, Huntington's disease or Parkinson'sdisease, but is preferably ischemic stroke, traumatic brain injury, orspinal cord injury. Further, the invention provides methods foradministering an additional active agent. The ligands of the inventionmay be administered in a pharmaceutical composition containing apharmaceutically acceptable excipient. The excipient may be suitable fororal administration. Thus, the composition may be in the form of atablet, a capsule, or a soft-gel capsule.

Alternatively, the excipient may be liquid suited to intravenous,intramuscular, or subcutaneous administration. Alternatively, theexcipient may be suited to transdermal administration, or buccaladministration. The sigma receptor ligand is preferably1′-[4-[1-(4-fluorophenyl)-1H-indol-3-yl]-1-butyl] spiro[isobenzofuran-1(3H, 4′-piperidine] (siramesine or Lu 28-179), or apharmaceutically acceptable salt, or solvate thereof.

The present invention provides methods and compositions for therehabilitation of patients with a central nervous system disorder, suchas stroke, spinal cord ischemia, spinal cord injury and traumatic braininjury. The invention is based on the discovery that sigma receptorligands, preferably siramesine, when administered to patients, withinabout 48 hours after a stroke, and for a period of one to three months,preferably administered up to one year, or more preferably, administeredcontinuously, allows the patients to recover from the dysfunctionalstate. The ligand can be delivered alone or in combination withadditional agents. Siramesine may be administered, for example, dailyover the course of the treatment.

Accordingly, in one aspect, the subject invention is directed to amethod for treating stroke in a subject, which comprises administeringto the subject a pharmaceutically effective amount of a ligand for thesigma receptor immediately after a stroke episode and for a period ofone to three months. The ligand for the sigma receptor is preferably1′-[4-[1-(4-fluorophenyl)-1H-indol-3-yl]-1-butyl] spiro[isobenzofuran-1(3H), 4′-piperidine] (siramesine or Lu 28-179), or apharmaceutically acceptable salt, or solvate thereof.

In another aspect of the invention, the administration of the sigmaligand to the subject commences not less than 24 hours, such as not lessthan 48 hours, one week, one month or three months, after aneurodegenerative disease, especially after an ischemic stroke,traumatic brain injury or spinal cord injury. From the start of thetreatment, the sigma ligand can be administered repeatedly, for exampledaily, for a period of, for example, one week, two weeks, one month,three months, one year or longer. For example the treatment can start atleast 24 hours, or at least 48 hours, at least one week after anischemic stroke, traumatic brain injury or spinal cord injury, andcontinue for one month, three months, six months or one year.

The treatment of the subject can be conducted under the direction of aphysician. In the course of the treatment, the physician may assess thesubject for evidence of neuronal regeneration. The evidence can beevidence of functional recovery or of a structural change in the brainor spinal cord. Thus, for example, the physician can measure one or morefunctional responses of the subject immediately prior to, or oncommencement of the treatment, and again after treatment. Thus,treatment can be continued until evidence of neuronal regeneration (orfunctional recovery) has been obtained.

As described in more detail hereinafter, the evidence of functionalrecovery may be, for example, recovery in a motor skill, cognitiveskill, speech or sensory perception and function. Particular mention maybe made of recovery in a motor skill and recovery in a cognitive skill.Evidence of neuronal regeneration may also be evidence of a structuralchange in the brain or spinal cord.

In another aspect of the invention, a packaged kit is provided for apatient to use in the treatment of a neurodegenerative disease tofacilitate neuronal regeneration (or functional recovery). The kitincludes a pharmaceutical formulation of siramesine, or salts orsolvates thereof, a container housing the pharmaceutical formulationduring storage and prior to administration, and instructions, e.g.,written instructions on a package insert or label, for carrying out drugadministration in a manner effective to treat the neurodegenerativedisease to facilitate neuronal regeneration (or functional recovery).The pharmaceutical formulation may be any formulation described herein,e.g., an oral dosage form containing a unit dosage of the ligand for thesigma receptor, the unit dosage being a therapeutically effective dosagefor treatment of the disease.

These and other aspects of the present invention will become evidentupon reference to the following detailed description. In addition,various references are set forth herein which describe in more detailcertain procedures or compositions, and are therefore incorporated byreference in their entirety.

DETAILED DESCRIPTION

I. Definitions

Unless otherwise stated, the following terms used in this application,including the specification and claims, have the definitions givenbelow. It must be noted that, as used in the specification and theappended claims, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise. Definition ofstandard chemistry terms may be found in reference works, includingCarey and Sundberg (1992) “Advanced Organic Chemistry 3^(rd) Ed.” Vols.A and B, Plenum Press, New York. The practice of the present inventionwill employ, unless otherwise indicated, conventional methods of massspectroscopy, protein chemistry, biochemistry, recombinant DNAtechniques and pharmacology, within the skill of the art.

The term “agonist” means a molecule such as a compound, a drug, anenzyme activator or a hormone that enhances the activity of anothermolecule or the activity of the sigma receptor site.

The term “antagonist” means a molecule such as a compound, a drug, anenzyme inhibitor, or a hormone, that diminishes or prevents the actionof another molecule or the activity of the sigma receptor site.

The term “stroke” broadly refers to the development of neurologicaldeficits associated with impaired blood flow to the brain regardless ofcause. Potential causes include, but are not limited to, thrombosis,hemorrhage and embolism. Thrombus, embolus, and systemic hypotension areamong the most common causes of cerebral ischemic episodes. Otherinjuries may be caused by hypertension, hypertensive cerebral vasculardisease, rupture of an aneurysm, an angioma, blood dyscrasias, cardiacfailure, cardiac arrest, cardiogenic shock, septic shock, head trauma,spinal cord trauma, seizure, bleeding from a tumor, or other blood loss.

By “ischemic episode” is meant any circumstance that results in adeficient supply of blood to a tissue. When the ischemia is associatedwith a stroke, it can be either global or focal ischemia, as definedbelow. The term “ischemic stroke” refers more specifically to a type ofstroke that is of limited extent and caused due to blockage of bloodflow. The term “ischemic stroke” includes cerebral ischemia aftercardiac arrest, stroke, and multi-infarct dementia, including thoseresulting from surgery. Cerebral ischemic episodes result from adeficiency in the blood supply to the brain. The spinal cord, which isalso a part of the central nervous system, is equally susceptible toischemia resulting from diminished blood flow.

By “focal ischemia,” as used herein in reference to the central nervoussystem, is meant the condition that results from the blockage of asingle artery that supplies blood to the brain or spinal cord, resultingin damage to the cells in the territory supplied by that artery.

By “global ischemia,” as used herein in reference to the central nervoussystem, is meant the condition that results from a general diminution ofblood flow to the entire brain, forebrain, or spinal cord, which causesthe death of neurons in selectively vulnerable regions throughout thesetissues. The pathology in each of these cases is quite different, as arethe clinical correlates. Models of focal ischemia apply to patients withfocal cerebral infarction, while models of global ischemia are analogousto cardiac arrest, and other causes of systemic hypotension.

By “neuroprotective agent” as used herein is meant a compound effectiveto reduce neuronal cell death, including the ability to inhibit thespread of neuronal damage from the initial site of injury.

The term “microarray” refers to an array of distinct polynucleotides oroligonucleotides synthesized or attached or deposited on a substrate,such as paper, nylon or other type of membrane, filter, chip, glassslide, beads, or any other suitable solid support, at a desired density.

The terms “effective amount” or “pharmaceutically effective amount”refer to a nontoxic but sufficient amount of the agent to provide thedesired biological result. That result can be reduction and/oralleviation of the signs, symptoms, or causes of a disease, or any otherdesired alteration of a biological system. For example, an “effectiveamount” for therapeutic uses is the amount of the composition comprisinga ligand for the sigma receptor disclosed herein required to provide aclinically significant decrease in neurodegenerative disease, such asthose resulting from ischemic stroke. An appropriate “effective” amountin any individual case may be determined by one of ordinary skill in theart using routine experimentation.

As used herein, the terms “treat” or “treatment” are usedinterchangeably and are meant to indicate a postponement of developmentof neurodegenerative diseases and/or a reduction in the severity of suchsymptoms that will or are expected to develop. The terms further includeameliorating existing neurodegenerative symptoms, preventing additionalsymptoms, and ameliorating or preventing the underlying metabolic causesof symptoms.

By “pharmaceutically acceptable” or “pharmacologically acceptable” ismeant a material which is not biologically or otherwise undesirable,i.e., the material may be administered to an individual without causingany undesirable biological effects or interacting in a deleteriousmanner with any of the components of the composition in which it iscontained.

By “physiological pH” or a “pH in the physiologically acceptable range”is meant a pH in the range of approximately 7.2 to 8.0 inclusive, moretypically in the range of approximately 7.2 to 7.6 inclusive.

As used herein, the term “subject” encompasses mammals and non-mammals.Examples of mammals include, but are not limited to, any member of theMammalian class: humans, non-human primates such as chimpanzees, andother apes and monkey species; farm animals such as cattle, horses,sheep, goats, swine; domestic animals such as rabbits, dogs, and cats;laboratory animals including rodents, such as rats, mice and guineapigs, and the like. Examples of non-mammals include, but are not limitedto, birds, fish and the like. The term does not denote a particular ageor gender.

The term “pharmaceutically acceptable salt” of a compound means a saltthat is pharmaceutically acceptable and that possesses the desiredpharmacological activity of the parent compound. Such salts, forexample, include:

-   -   (1) acid addition salts, formed with inorganic acids such as        hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,        phosphoric acid, and the like; or formed with organic acids such        as acetic acid, propionic acid, hexanoic acid,        cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic        acid, malonic acid, succinic acid, malic acid, maleic acid,        fumaric acid, tartaric acid, citric acid, benzoic acid,        3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid,        methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic        acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid,        2-naphthalenesulfonic acid,        4-methylbicyclo-[2.2.2]oct-2-ene-1-carboxylic acid,        glucoheptonic acid,        4,4′-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid),        3-phenylpropionic acid, trimethylacetic acid, tertiary        butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic        acid, hydroxynaphthoic acid, salicylic acid, stearic acid,        muconic acid, and the like;    -   (2) salts formed when an acidic proton present in the parent        compound either is replaced by a metal ion, e.g., an alkali        metal ion, an alkaline earth ion, or an aluminum ion; or        coordinates with an organic base. Acceptable organic bases        include ethanolamine, diethanolamine, triethanolamine,        tromethamine, N-methylglucamine, and the like. Acceptable        inorganic bases include aluminum hydroxide, calcium hydroxide,        potassium hydroxide, sodium carbonate, sodium hydroxide, and the        like. It should be understood that a reference to a        pharmaceutically acceptable salt includes the solvent addition        forms or crystal forms thereof, particularly solvates or        polymorphs. Solvates contain either stoichiometric or        non-stoichiometric amounts of a solvent, and are often formed        during the process of crystallization. Hydrates are formed when        the solvent is water, or alcoholates are formed when the solvent        is alcohol. Polymorphs include the different crystal packing        arrangements of the same elemental composition of a compound.        Polymorphs usually have different X-ray diffraction patterns,        infrared spectra, melting points, density, hardness, crystal        shape, optical and electrical properties, stability, and        solubility. Various factors such as the recrystallization        solvent, rate of crystallization, and storage temperature may        cause a single crystal form to dominate.

The term “optional” or “optionally” means that the subsequentlydescribed event or circumstance may or may not occur, and that thedescription includes instances where the event or circumstance occursand instances where it does not. For example, the phrase “optionallyanother drug” means that the patient may or may not be given a drugother than the sigma receptor ligands. “Another drug” as used herein ismeant any chemical material or compound suitable for administration to amammalian, preferably human, which induces a desired local or systemiceffect. In general, this includes: anorexics; anti-infectives such asantibiotics and antiviral agents, including many penicillins andcephalosporins; analgesics and analgesic combinations; antiarrhythmics;antiarthritics; antiasthmatic agents; anticholinergics; anticonvulsants;antidiabetic agents; antidiarrheals; antihelminthics; antihistamines;antiinflammatory agents; antimigraine preparations; antinauseants;antineoplastics; antiparkinsonism drugs; antipruritics; antipsychotics;antipyretics; antisense agents; antispasmodics; cardiovascularpreparations including calcium channel blockers and beta-blockers suchas pindolol; antihypertensives; central nervous system stimulants; coughand cold preparations, including decongestants; diuretics;gastrointestinal drugs, including H₂-receptor antagonists;sympathomimetics; hormones such as estradiol and other steroids,including corticosteroids; hypnotics; immunosuppressives; musclerelaxants; parasympatholytics; psychostimulants; sedatives;tranquilizers; thrombolytics; neuroprotectants; radical scavengers andvasodilators.

The term “functional rehabilitation” as used herein means physicaltherapy, occupational therapy, and the like. It is anticipated that drugtreatment will commence with rehabilitation.

II. Sigma Receptor

The Sigma Receptor was identified from a microarray analysis of anenriched environment experiment. The invention further provides methodsfor the identification of compounds that modulate the expression of thesigma receptor for the treatment of central nervous system disorders andfor stimulating nerve cell survival and regeneration in subjects withneurodegenerative disorders. The microarray analysis identified genesthat are differentially expressed after cortical ischemia andpostischemic environmental enrichment brain tissue, relative to theirexpression in normal, or non-enriched environment are identified anddescribed

Further, the invention provides methods of treating a subject exhibitingchanges in the above gene expression, wherein the therapeuticintervention results in cell genesis and an enhanced subsequentfunctional recovery in brain. The inventors have found that sigmareceptors expression decrease in the vulnerable regions after middlecerebral artery occlusion (MCAO) in standard conditions and increaseafter MCAO when the subject is exposed to conditions of enrichedenvironment. An increase has also been detected after MCAO in theresistant regions of the brain. Thus, for a subject suffering from focalor global ischemia of the brain, sigma receptor ligands are administeredafter the insult, and for a period of time sufficient to facilitatefunctional recovery. The pharmaceutical intervention leads to fasterfunctional recovery.

In one aspect, an array or a micro array can be used to obtain the geneexpression of interest. Typically, probe oligonucleotides areimmobilized on a solid support, and then contacted with a samplecontaining labeled target oligonucleotides under hybridizationconditions to produce a hybridization pattern. After hybridization, thefluorescence or radioactivity, such as ³³P utilized for in situhybridization, measurements are analyzed to determine the level ofhybridization of the targets to the probes. The information is useful indetermining gene function, gene-splicing, understanding the geneticbasis of disease, diagnosing disease, in developing and monitoring theactivity of therapeutic agents, detecting the presence or absence of apolymorphism, and the like (Heller, R. et al. (1997) Proc. Natl. Acad.Sci. 94:2150-55). The probe and target oligonucleotides can be obtainedfrom the RNA or DNA of a biological sample. The oligonucleotides willgenerally be a DNA that has been reverse-transcribed from RNA derivedusually from a naturally occurring source, where the RNA can be totalRNA, PolyA+mRNA, amplified RNA and the like. The initial mRNA sample maybe derived from a physiological source including a single-celledorganism such as yeast, from a eukaryotic source, or a multicellularorganism including plants and animals, particularly mammals and organs,tissues, and cells derived from the mammals such as from any bodilyfluids (such as blood, urine, saliva, phlegm, gastric juices, etc.),cultured cells, biopsies, or other tissue preparations. Methods ofisolating RNA from cells, tissues, organs or whole organisms are knownto those of skill in the art and are described in Sambrook, Fritsch &Maniatis (1989) Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Press).). In particular, RNA from the brain, such as from medial,rostral, frontal, hippocampus and striatum regions of the subject arepurified and cloned for use in the microarray experiments.

The hybridization pattern can be used to determine quantitativeinformation about the genetic profile of the nucleic acids in the samplethat was contacted with the array to generate the hybridization pattern,as well as the physiological source from which the labeled samplenucleic acid was derived. The data provides information about thephysiological source from which the sample nucleic acids were derived,such as the types of genes expressed in the tissue or cell which is thephysiological source, as well as the levels of expression of each gene,particularly in quantitative terms.

It was discovered that rats subjected to MCAO and then exposed to anenriched environment demonstrated upregulation of the type 1 sigmareceptor mRNA in striatum and frontal cortex and a downregulation of thereceptor in medial cortex. The frontal cortex has been implicated incontrol of sensory-motor function. Hence the role of pharmacologicalinterventions in the postischemic rehabilitation phase. Thus, theadministration of sigma receptor ligands, such as sigma receptoragonists, can improve functional recovery of the subject following astroke. Further, the interactions between drugs and environmentalfactors, such as an enriched environment, can be used to improvefunctional recovery.

Without wishing to be bound by theory, it is believed that sigma ligandsmay facilitate neuronal regeneration and functional recovery bymimicking the effects of an enriched or stimulating environment.

III. Sigma Receptor Ligands

The sigma receptor ligands can be used in methods and compositions fortreating neurodegenerative diseases, and for improving functionalrecovery from neurodegenerative diseases.

Several ligands for the sigma receptor are known which may find use withthe subject methods. For example, Manallack, D. T. et al., Eur. J.Pharmacol., 144: 231-235 (1987), disclose phencyclidine compounds thathave affinity for the sigma binding sites, and that the sigma siteaffinity was shown to be enhanced by large N-alkyl substituents, e.g.,benzyl or phenylethyl. Largent, B. L. et al., Mol. Pharmacol., 32:772-784 (1987), teach that several piperidine and piperazine derivativeshave sigma receptor activity, and suggest that compounds containing morelipophilic substituents afford greater affinity for the sigma receptorbinding sites. Cocaine-related compounds were shown to have sigmareceptor binding activity by Sharkey, J. et al., Eur. J. Pharmacol.,149: 171-174 (1988). European Patent Application 362,001 describesα,α-disubstituted N-cycloalkylalkylamines having specific affinity forsigma receptors and European Patent Application 445,013 describesN-cycloalkylalkylamines having specific affinity for sigma receptors.The sigma receptor ligands described in both of these Europeanapplications are useful in the treatment of psychoses andgastrointestinal complaints. PCT publication WO 91/03243 includes adescription of 1-cycloalkylpiperidines having specific antagonistactivity toward sigma receptors and which are useful in the treatment ofpsychoses and dyskinesias. PCT publication WO 93/09094 includes adescription of ethers derived from alkyl piperidines or pyrrolidineswhich are antipsychotic agents. Additional substituted piperidines andpiperazines that are sigma receptor ligands are disclosed in the PCTpublication WO 94/24116. The sigma receptor affinities of1,4-(diphenylalkyl)piperazine derivatives and their use for cerebralfunction disorders such as dementia, depression and schizophrenia aredescribed in U.S. Pat. No. 5,736,546. U.S. Pat. No. 6,087,346 disclosescertain phenylalkyl-amine, aminotetralin, piperazine, piperidine andrelated compounds bind to the sigma receptor, and can be used for thefor the treatment of central nervous system disorders, neurologicaldisorders, gastrointestinal disorders, drug abuse, angina, migraine,hypertension and depression. Other sigma receptor ligands includeBMY-14802 caramiphen and haloperidol that were found to have in vivoprotective effects against NMDA-induced toxicity and seizures (M.Pontecorvo et al., (1991) Brain Res. Bull., 26:461-465). Additionalsigma receptor ligands include, for example, 3PP-HCl, haloperidol,allyl-normetazocine (also called SKF 10047), normetazocine, U-50488tartrate, carbetapentane, cyclazocine, ifenprodil, DTG(1,3-Di 2tolylguanidine), L693,409, PTPP, 4PPBP (4-phenyl-1-(4-phenylbutyl)piperidinemaleate), BD 1063, IPAB iodobenzamide, SM-21, BD1008.

IV. Methods for Identifying Sigma Receptor Ligands

Methods for identifying compounds that are sigma receptor ligands areknown in the art. One method used to identify compounds that are ligandsfor the sigma receptor involves placing cells, tissues, or preferably acellular extract or other preparation containing sigma receptors incontact with several known concentrations of a test compound in a buffercompatible with receptor activity, and assaying for ligand bindingand/or receptor activity. The method can be performed eithersequentially or in a multiplexed format. The use of in vitro bindingassays with known specific ligands can allow for the determination ofligand affinities for sigma 1 or sigma 2 receptors as described in LangaF. (2003) Eur. J. Neuroscience, 18:2188-2196. Other methods fordetermining compounds that are ligands for the sigma receptor can beemployed as will be apparent to those of skill in the art based on thedisclosure herein.

The sigma ligand is preferably siramesine (compound V below) or itssalts, or solvates. However, all of the following compounds are sigmareceptor ligands:

In another method, rational drug design, based upon structural studiesof the molecular shapes of the sigma receptor ligands identified aboveand known ligands or analogs may be used to identify compounds whosethree-dimensional structure is complementary to that of the active siteof the sigma receptors. These compounds may be determined by a varietyof techniques, including molecular mechanics calculations, moleculardynamics calculations, constrained molecular dynamics calculations inwhich the constraints are determined by NMR spectroscopy, distancegeometry in which the distance matrix is partially determined by NMRspectroscopy, x-ray diffraction, or neutron diffraction techniques. Inthe case of all these techniques, the structure can be determined in thepresence or absence of any ligands known to interact with sigmareceptors.

The sigma receptor ligands thus identified or designed can besubsequently tested for their ability to treat and/or preventneurodegenerative diseases. In one method, the compounds are tested fortheir ability to modulate the sigma receptors, such as, for example,sigma-1 (accession numbers NM_(—)005866, NM_(—)147157, NM_(—)147158,NM_(—)147159, and NM_(—)147160), sigma-2, or recombinant sigmareceptors. Lead compounds identified during these screens can serve asthe basis for the synthesis of more active analogs. Lead compoundsand/or active analogs generated therefrom can be formulated intopharmaceutical compositions effective in treating neurological disorderssuch as stroke, epilepsy and neurodegenerative disorders.

V. Synthesis of the Sigma Receptor Ligands

Some sigma receptor ligands are commercially available. Methods ofpreparing many are described in the patent and scientific literature,for example fluvoxamine (U.S. Pat. No. 4,085,225), 4-IBD (John et. al.(1999) Nuclear Medicine & Biology 26:377-382), Pre-084 (U.S. Pat. No.5,223,530), SA4503 (U.S. Pat. No. 5,736,546), siramesine (US 5,665,725),OPC-14523 (U.S. Pat. No. 5,556,857), BD-737 (U.S. Pat. No. 5,130,330 andU.S. Pat. No. 5,739,158), Igmesine (U.S. Pat. No. 5,034,419).

VI. Neuronal Regeneration and Functional Recovery

In one aspect of the invention, methods of treating a subject areprovided wherein the sigma receptor ligands I-IX, or salts or solvatesthereof are administered after stroke and for a sufficient period oftime necessary for treatment, such as from about 1 week to about 1 monthor to about 12 months or administered continuously until the desiredtherapeutic effect is observed. Preferably, the sigma receptor ligand issiramesine, or salts or solvates thereof. In another aspect of theinvention methods of treating a subject are provided wherein the sigmareceptor ligand siramesine, or salts or solvates thereof is administeredafter stroke and for a sufficient period of time necessary fortreatment, such as from about 1 week to about 1 month or to about 12months or administered continuously until the desired therapeutic effectis observed and wherein the subject is also exposed to a rich,stimulating environment, such as an enriched environment and tofunctional rehabilitation, so that functional recovery of the patientfrom the adverse consequences of the central nervous system injury isimproved.

Functional recovery occurs when the functions of a damaged region ofneural tissue is taken over by other areas that normally did notpreviously play a role in that particular function and the changes inthe neural function lead to changes in behavior or in the capacity forbehavior. Functional recovery is also referred to as neural plasticity.Functional recovery in the brain thus refers to functional andstructural reorganization, upregulation or downregulation of a neuralresponse to an event, and the establishment of new functional andstructural connections by means of collateral sprouting and compensatorysynaptogenesis as well as neurogenesis.

An improvement in the functional recovery of the patient can beassessed, for example, by using functional/behavioral tests to assesssensorimotor and reflex function of the patient's motor skills, such asposture, balance, grasp, or gait, cognitive skills, speech, and/orsensory perception and function including visual ability, taste,olfaction, and proprioception improve as a result of administrating thesigma receptor ligands according to the invention. In another aspect,functional recovery of the patient can be determined by histologicalanalysis that includes determining the length of the axonal bundles, anincrease in the neuronal regeneration at the site of injury, evaluatingthe dendritic morphology and the number of spines, and the like. In yetanother aspect, the improvement in the functional recovery of thepatient can be determined by using non-invasive techniques thatdetermine structural alterations in the brain that lead to changes inneural function. Thus, electrophysiological (electroencephalograph (EEG)or evoked response potential (ERP)), electromyographic (EMG),neurochemical (CSF metabolites), peripheral (circulating beta-endorphinlevels), radiological (CT scan, MRI) and clinical (Pupillary LightReflex, posture, taste) measures can be used to measure functionalrecovery of the patients. In addition, the above techniques can be usedto select patients who may be likely to successfully respond to thetreatment of the invention.

The sigma receptor ligand siramesine is administered to mimic theeffects of an enriched or stimulating environment. It is known thatpost-ischemic housing in an enriched or stimulating environment canimprove functional outcome after brain ischemia in the rat. After anexperimental brain infarction the rats housed in an enriched environmentwith the opportunity for various activities and interaction with otherrats did better than rats housed in standard laboratory environment. Anenriched environment that allowed for free physical activity combinedwith social interaction resulted in the best performance without changein infarct volume. An enriched environment may stimulate mechanisms thatenhance brain plasticity after focal brain ischemia. It has been shownthat housing rats in a stimulating environment significantly increasesspine density in superficial cortical layers in intact and lesionedbrain.

In one aspect of the invention, the stimulating environment comprisessocial interaction, motor activity, electrical stimulation of the brain,a change in the habitation, and the like. For example, the subject canbe encouraged to use an impaired limb to improve sensorimotor function,may be subjected to daily physical routines, such as walking,stretching, weight lifting, and the like, or encouraged to play games,such as baseball, hockey, soccer, or board games. In addition, thedomicile of the subject may be changed to stimulate brain activity, suchas by changing the colors in the room, providing textured material,providing items manufactured from different materials, such as wood,steel, and the like. The knowledge to customize the stimulatingenvironment for a particular patient is known to those in thephysiotherapy and occupational therapy arts.

In another aspect, the stimulating environment comprises directstimulation of the brain or a region of the brain. For example,electrical impulses can be applied to the brain as described in U.S.Pat. Nos. 6,339,725, and 5,611,350, and other methods known in the art.Alternatively, the brain or a region of the brain can be stimulated bylocalized administration of drugs, such as acetylcholine, nerve growthfactors, such nerve stimulating agents, neuronal or glial growth factorsand other neuronal modulating drugs.

As one of skill in the art will recognize, the timing of administeringthe dosage containing the sigma ligands can vary. In one aspect of theinvention, the sigma receptor ligands are administered after a stroke.The administration of the ligands can be initiated within the first weekof the onset of the symptoms, preferably at least 24 hours, or at least48 hours of the onset of the symptoms. In another aspect of theinvention, the sigma receptor ligands are administered to the patientconcurrently with exposure to a stimulating environment. Preferably, thesigma receptor ligands are administered after a stroke at a time whenthe patient is subjected to a stimulating environment, and the ligandsare administered for about 1 month to about 3 months to facilitatefunctional recovery. Preferably, the ligands, and compositionscomprising the ligands are administered up to about 12 months or longer,or, even more preferably, administered continuously.

VII. Pharmaceutical Formulations and Modes of Administration

The methods described herein use pharmaceutical compositions comprisingthe molecules described above, where the molecule is preferablysiramesine, together with one or more pharmaceutically acceptableexcipients or vehicles, and optionally other therapeutic and/orprophylactic ingredients. Such excipients include liquids such as water,saline, glycerol, polyethyleneglycol, hyaluronic acid, ethanol, etc.Suitable excipients for non-liquid formulations are also known to thoseof skill in the art. Pharmaceutically acceptable salts can be used inthe compositions of the present invention and include, for example,mineral acid salts such as hydrochlorides, hydrobromides, phosphates,sulfates, and the like; and the salts of organic acids such as acetates,propionates, malonates, benzoates, and the like. A thorough discussionof pharmaceutically acceptable excipients and salts is available inRemington's Pharmaceutical Sciences, 18th Edition (Easton, Pa.: MackPublishing Company, 1990).

Additionally, auxiliary substances, such as wetting or emulsifyingagents, biological buffering substances, surfactants, and the like, maybe present in such vehicles. A biological buffer can be virtually anysolution which is pharmacologically acceptable and which provides theformulation with the desired pH, i.e., a pH in the physiologicallyacceptable range. Examples of buffer solutions include saline, phosphatebuffered saline, Tris buffered saline, Hank's buffered saline, and thelike.

Depending on the intended mode of administration, the pharmaceuticalcompositions may be in the form of solid, semi-solid or liquid dosageforms, such as, for example, tablets, suppositories, pills, capsules,powders, liquids, suspensions, creams, ointments, lotions or the like,preferably in unit dosage form suitable for single administration of aprecise dosage. The compositions will include an effective amount of theselected drug in combination with a pharmaceutically acceptable carrierand, in addition, may include other pharmaceutical agents, adjuvants,diluents, buffers, etc.

The invention includes a pharmaceutical composition comprising acompound of the present invention including isomers, racemic ornon-racemic mixtures of isomers, or pharmaceutically acceptable salts orsolvates thereof together with one or more pharmaceutically acceptablecarriers, and optionally other therapeutic and/or prophylacticingredients.

In general, the compounds of this invention will be administered in atherapeutically effective amount by any of the accepted modes ofadministration. Suitable dosage ranges depend upon numerous factors suchas the severity of the disease to be treated, the age and relativehealth of the subject, the potency of the compound used, the route andform of administration, the indication towards which the administrationis directed, and the preferences and experience of the medicalpractitioner involved. One of ordinary skill in the art of treating suchdiseases will be able, without undue experimentation and in relianceupon personal knowledge and the disclosure of this application, toascertain a therapeutically effective amount of the compounds of thisinvention for a given disease.

In general, compounds of this invention will be administered aspharmaceutical formulations including those suitable for oral (includingbuccal and sub-lingual), rectal, nasal, topical, pulmonary, vaginal orparenteral (including intramuscular, intraarterial, intrathecal,subcutaneous and intravenous) administration or in a form suitable foradministration by inhalation or insufflation. The preferred manner ofadministration is intravenous or oral using a convenient daily dosageregimen which can be adjusted according to the degree of affliction.

For solid compositions, conventional nontoxic solid carriers include,for example, pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose,magnesium carbonate, and the like. Liquid pharmaceutically administrablecompositions can, for example, be prepared by dissolving, dispersing,etc., an active compound as described herein and optional pharmaceuticaladjuvants in an excipient, such as, for example, water, saline, aqueousdextrose, glycerol, ethanol, and the like, to thereby form a solution orsuspension. If desired, the pharmaceutical composition to beadministered may also contain minor amounts of nontoxic auxiliarysubstances such as wetting or emulsifying agents, pH buffering agentsand the like, for example, sodium acetate, sorbitan monolaurate,triethanolamine sodium acetate, triethanolamine oleate, etc. Actualmethods of preparing such dosage forms are known, or will be apparent,to those skilled in this art; for example, see Remington'sPharmaceutical Sciences, referenced above.

For oral administration, the composition will generally take the form ofa tablet, capsule, a softgel capsule or may be an aqueous or nonaqueoussolution, suspension or syrup. Tablets and capsules are preferred oraladministration forms. Tablets and capsules for oral use will generallyinclude one or more commonly used carriers such as lactose and cornstarch. Lubricating agents, such as magnesium stearate, are alsotypically added. Typically, the compounds of the invention can becombined with an oral, non-toxic, pharmaceutically acceptable, inertcarrier such as lactose, starch, sucrose, glucose, methyl callulose,magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol,sorbitol and the like. Moreover, when desired or necessary, suitablebinders, lubricants, disintegrating agents, and coloring agents can alsobe incorporated into the mixture. Suitable binders include starch,gelatin, natural sugars such as glucose or beta-lactose, cornsweeteners, natural and synthetic gums such as acacia, tragacanth, orsodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, andthe like. Lubricants used in these dosage forms include sodium oleate,sodium stearate, magnesium stearate, sodium benzoate, sodium acetate,sodium chloride, and the like. Disintegrators include, withoutlimitation, starch, methyl cellulose, agar, bentonite, xanthan gum, andthe like.

Thus, for example, capsules can be prepared by conventional proceduresso that the dosage unit is 100 mg of the compounds of the invention, 100mg of cellulose and 10 mg of magnesium stearate. A large number of unitcapsules may also prepared by filling standard two-piece hard gelatincapsules each with 100 mg of powdered active ingredient, 150 mg oflactose, 50 mg of cellulose, and 10 mg magnesium stearate. Or, tabletsmay be prepared by conventional procedures so that the dosage unit is100 mg of the compounds of the invention, 150 mg of lactose, 50 mg ofcellulose and 10 mg of magnesium stearate. A large number of tablets mayalso be prepared by conventional procedures such that the dosage unitwas 100 mg of the compounds of the invention, and other ingredients canbe 0.2 mg of colloidal silicon dioxide, 5 mg of magnesium stearate, 250mg of microcrystalline cellulose, 10 mg of starch and 100 mg of lactose.Appropriate coatings may be applied to increase palatability or delayabsorption.

When liquid suspensions are used, the active agent may be combined withany oral, non-toxic, pharmaceutically acceptable inert carrier such asethanol, glycerol, water, and the like and with emulsifying andsuspending agents. If desired, flavoring, coloring and/or sweeteningagents may be added as well. Other optional components for incorporationinto an oral formulation herein include, but are not limited to,preservatives, suspending agents, thickening agents, and the like.

Parenteral formulations can be prepared in conventional forms, either asliquid solutions or suspensions, solid forms suitable for solubilizationor suspension in liquid prior to injection, or as emulsions. Preferably,sterile injectable suspensions are formulated according to techniquesknown in the art using suitable carriers, dispersing or wetting agentsand suspending agents. The sterile injectable formulation may also be asterile injectable solution or a suspension in a nontoxic parenterallyacceptable diluent or solvent. Among the acceptable vehicles andsolvents that may be employed are water, Ringer's solution and isotonicsodium chloride solution. In addition, sterile, fixed oils, fatty estersor polyols are conventionally employed as solvents or suspending media.In addition, parenteral administration may involve the use of a slowrelease or sustained release system such that a constant level of dosageis maintained.

Parenteral administration includes intraarticular, intravenous,intramuscular, intradermal, intraperitoneal, and subcutaneous routes,and include aqueous and non-aqueous, isotonic sterile injectionsolutions, which can contain antioxidants, buffers, bacteriostats, andsolutes that render the formulation isotonic with the blood of theintended recipient, and aqueous and non-aqueous sterile suspensions thatcan include suspending agents, solubilizers, thickening agents,stabilizers, and preservatives. Administration via certain parenteralroutes can involve introducing the formulations of the present inventioninto the body of a patient through a needle or a catheter, propelled bya sterile syringe or some other mechanical device such as an continuousinfusion system. A formulation provided by the present invention can beadministered using a syringe, injector, pump, or any other devicerecognized in the art for parenteral administration.

Preferably, sterile injectable suspensions are formulated according totechniques known in the art using suitable carriers, dispersing orwetting agents and suspending agents. The sterile injectable formulationcan also be a sterile injectable solution or a suspension in a nontoxicparenterally acceptable diluent or solvent. Among the acceptablevehicles and solvents that can be employed are water, Ringer's solutionand isotonic sodium chloride solution. In addition, sterile, fixed oils,fatty esters or polyols are conventionally employed as solvents orsuspending media. In addition, parenteral administration can involve theuse of a slow release or sustained release system such that a constantlevel of dosage is maintained.

Preparations according to this invention for parenteral administrationinclude sterile aqueous or non-aqueous solutions, suspensions, oremulsions. Examples of non-aqueous solvents or vehicles are propyleneglycol, polyethylene glycol, vegetable oils, such as olive oil and cornoil, gelatin, and injectable organic esters such as ethyl oleate. Suchdosage forms can also contain adjuvants such as preserving, wetting,emulsifying, and dispersing agents. They can be sterilized by, forexample, filtration through a bacteria retaining filter, byincorporating sterilizing agents into the compositions, by irradiatingthe compositions, or by heating the compositions. They can also bemanufactured using sterile water, or some other sterile injectablemedium, immediately before use.

The formulations can optionally contain an isotonicity agent. Theformulations preferably contain an isotonicity agent, and glycerin isthe most preferred isotonicity agent. The concentration of glycerin,when it is used, is in the range known in the art, such as, for example,about 1 mg/mL to about 20 mg/mL.

The pH of the parenteral formulations can be controlled by a bufferingagent, such as phosphare, acetate, TRIS or L-arginine. The concentrationof the buffering agent is preferably adequate to provide buffering ofthe pH during storage to maintain the pH at a target pH±0.2 pH unit. Thepreferred pH is between about 7 and about 8 when measured at roomtemperature.

Other additives, such as a pharmaceutically acceptable solubilizers likeTween 20® (polyoxyethylene (20) sorbitan monolaurate), Tween 40®(polyoxyethylene (20) sorbitan monopalmitate), Tween 80®(polyoxyethylene (20) sorbitan monooleate), Pluronic F68®(polyoxyethylene polyoxypropylene block copolymers), and PEG(polyethylene glycol) can optionally be added to the formulation, andmay be useful if the formulations will contact plastic materials. Inaddition, the parenteral formulations can contain various antibacterialand antifungal agents, for example, parabens, chlorobutanol, phenol,sorbic acid, thimerosal, and the like.

Sterile injectable solutions are prepared by incorporating one or moreof the compounds of the invention in the required amount in theappropriate solvent with various of the other ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the various sterilized activeingredients into a sterile vehicle which contains the basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum-drying andfreeze-drying techniques which yield a powder of the active ingredientplus any additional desired ingredient from a previouslysterile-filtered solution thereof. Thus, for example, a parenteralcomposition suitable for administration by injection is prepared bystirring 1.5% by weight of active ingredient in 10% by volume propyleneglycol and water. The solution is made isotonic with sodium chloride andsterilized.

Alternatively, the pharmaceutical compositions of the invention may beadministered in the form of suppositories for rectal administration.These can be prepared by mixing the agent with a suitable nonirritatingexcipient which is solid at room temperature but liquid at the rectaltemperature and therefore will melt in the rectum to release the drug.Such materials include cocoa butter, beeswax and polyethylene glycols.

The pharmaceutical compositions of the invention may also beadministered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, propellants such as fluorocarbons or nitrogen, and/orother conventional solubilizing or dispersing agents.

Preferred formulations for topical drug delivery are ointments andcreams. Ointments are semisolid preparations which are typically basedon petrolatum or other petroleum derivatives. Creams containing theselected active agent, are, as known in the art, viscous liquid orsemisolid emulsions, either oil-in-water or water-in-oil. Cream basesare water-washable, and contain an oil phase, an emulsifier and anaqueous phase. The oil phase, also sometimes called the “internal”phase, is generally comprised of petrolatum and a fatty alcohol such ascetyl or stearyl alcohol; the aqueous phase usually, although notnecessarily, exceeds the oil phase in volume, and generally contains ahumectant. The emulsifier in a cream formulation is generally anonionic, anionic, cationic or amphoteric surfactant. The specificointment or cream base to be used, as will be appreciated by thoseskilled in the art, is one that will provide for optimum drug delivery.As with other carriers or vehicles, an ointment base should be inert,stable, nonirritating and nonsensitizing.

Formulations for buccal administration include tablets, lozenges, gelsand the like. Alternatively, buccal administration can be effected usinga transmucosal delivery system as known to those skilled in the art. Thecompounds of the invention may also be delivered through the skin ormuscosal tissue using conventional transdermal drug delivery systems,i.e., transdermal “patches” wherein the agent is typically containedwithin a laminated structure that serves as a drug delivery device to beaffixed to the body surface. In such a structure, the drug compositionis typically contained in a layer, or “reservoir,” underlying an upperbacking layer. The laminated device may contain a single reservoir, orit may contain multiple reservoirs. In one embodiment, the reservoircomprises a polymeric matrix of a pharmaceutically acceptable contactadhesive material that serves to affix the system to the skin duringdrug delivery. Examples of suitable skin contact adhesive materialsinclude, but are not limited to, polyethylenes, polysiloxanes,polyisobutylenes, polyacrylates, polyurethanes, and the like.Alternatively, the drug-containing reservoir and skin contact adhesiveare present as separate and distinct layers, with the adhesiveunderlying the reservoir which, in this case, may be either a polymericmatrix as described above, or it may be a liquid or gel reservoir, ormay take some other form. The backing layer in these laminates, whichserves as the upper surface of the device, functions as the primarystructural element of the laminated structure and provides the devicewith much of its flexibility. The material selected for the backinglayer should be substantially impermeable to the active agent and anyother materials that are present.

A pharmaceutically or therapeutically effective amount of thecomposition will be delivered to the subject. The precise effectiveamount will vary from subject to subject and will depend upon thespecies, age, the subject's size and health, the nature and extent ofthe condition being treated, recommendations of the treating physician,and the therapeutics or combination of therapeutics selected foradministration. Thus, the effective amount for a given situation can bedetermined by routine experimentation. For purposes of the presentinvention, generally a therapeutic amount will be in the range of about0.01 mg/kg to about 40 mg/kg body weight, more preferably about 0.1mg/kg to about 10 mg/kg, in at least one dose. In larger mammals theindicated daily dosage can be from about 1 mg to 300 mg, one or moretimes per day, more preferably in the range of about 10 mg to 200 mg.The subject may be administered as many doses as is required to reduceand/or alleviate the signs, symptoms, or causes of the disorder inquestion, or bring about any other desired alteration of a biologicalsystem.

The compounds of the present invention may be formulated for aerosoladministration, particularly to the respiratory tract and includingintranasal administration. The compound will generally have a smallparticle size for example of the order of 5 microns or less. Such aparticle size may be obtained by means known in the art, for example bymicronization. The active ingredient is provided in a pressurized packwith a suitable propellant such as a chlorofluorocarbon (CFC) forexample dichlorodifluoromethane, trichlorofluoromethane, ordichlorotetrafluoroethane, carbon dioxide or other suitable gas. Theaerosol may conveniently also contain a surfactant such as lecithin. Thedose of drug may be controlled by a metered valve. Alternatively theactive ingredients may be provided in a form of a dry powder, forexample a powder mix of the compound in a suitable powder base such aslactose, starch, starch derivatives such as hydroxypropylmethylcellulose and polyvinylpyrrolidine (PVP). The powder carrier will form agel in the nasal cavity. The powder composition may be presented in unitdose form for example in capsules or cartridges of e.g., gelatin orblister packs from which the powder may be administered by means of aninhaler.

When desired, formulations can be prepared with enteric coatings adaptedfor sustained or controlled release administration of the activeingredient.

The pharmaceutical preparations are preferably in unit dosage forms. Insuch form, the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, such as packeted tablets, capsules, and powders in vials orampoules. Also, the unit dosage form can be a capsule, tablet, cachet,or lozenge itself, or it can be the appropriate number of any of thesein packaged form.

VIII. Kits

In another aspect, the invention relates to pharmaceutical compositionsin kit form. The kit comprises container means for containing thecompositions such as a bottle, a foil packet, or another type ofcontainer. Typically the kit further comprises directions for theadministration of the compositions.

In another aspect, a packaged kit is provided that contains thepharmaceutical formulation to be administered, i.e., a pharmaceuticalformulation containing a ligand for the sigma receptor for the treatmentof a neurodegenerative disease to facilitate neuronal regeneration orfunctional recovery, a container, preferably sealed, for housing theformulation during storage and prior to use, and instructions forcarrying out drug administration in a manner effective to treat thedisease. The instructions will typically be written instructions on apackage insert and/or on a label. The formulation may be any suitableformulation as described herein. For example, the formulation may be anoral dosage form containing a unit dosage of the ligand for the sigmareceptor. The kit may contain multiple formulations of different dosagesof the same agent. The kit may also contain multiple formulations ofdifferent active agents.

For example, the kits for parenteral administration can comprise a) apharmaceutical composition comprising siramesine described above and apharmaceutically acceptable carrier, vehicle or diluent; and,optionally, b) instructions describing a method of using thepharmaceutical composition for treating or preventing the disease. Thekits can further include a device for administering the formulation(e.g., a syringe, a catheter, and the like). The kits for oraladministration can comprise the dosage formulation contained within acontainer, such as, for example, a paper or cardboard box, a glass orplastic bottle or jar, a re-sealable bag, or a blister pack withindividual doses. Blister packs generally consist of a sheet ofrelatively stiff material covered with a foil of a preferablytransparent plastic material. During the packaging process, havingrecesses the size and shape of the tablets or capsules, are formed inthe plastic foil. Subsequently, the tablets or capsules are placed inthe recesses and the sheet of relatively stiff material is sealedagainst the plastic foil at the face of the foil which is opposite fromthe direction in which the recesses were formed. As a result, thetablets or capsules are individually sealed. Preferably the strength ofthe sheet is such that the tablets or capsules can be removed from theblister pack by manually applying pressure on the recesses whereby anopening is formed in the sheet at the place of the recess. The tablet orcapsule can then be removed via the opening.

It may be desirable to provide a memory aid on the kit, e.g., in theform of numbers next to the tablets or capsules whereby the numberscorrespond with the days of the regimen which the dosage form sospecified should be administered. Another example of such a memory aidis a calendar printed on the card e.g., as follows “First Week, Monday,Tuesday, . . . etc. . . . Second Week, Monday, Tuesday, . . . ” etc.Other variations of memory aids will be readily apparent, such as, forexample, a mechanical counter which indicates the number of daily dosesthat has been dispensed, a microchip memory coupled with a liquidcrystal readout, or audible reminder signal which, for example, readsout the date that the last daily dose has been taken and/or reminds onewhen the next dose is to be taken, and the like.

EXAMPLES

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

Example 1

Animal Models for Neuronal Regeneration (Functional Recovery)

Male 3 months old SHR (spontaneous hypertensive) rats are used forinduction of stroke by MCA occlusion. This is the preferred strain sincemost stroke patients are hypertensive. The animals are anesthetized withMethohexital and a small craniectomy is made above the zygmotic arch toexpose the middle cerebral artery, which is occluded with a 10-0monofilament nylon thread distal to the origin of the striatal branches.The rats are not intubated and no catheters are inserted. Following MCAocclusion a large and reproducible infarct is obtained, leading to arobust sensorimotor deficit. The animals are kept on a 6 hr light/18 hdark cycle with free access to food and water. At two days after theMCAO the rats are treated with the compound I, II, III, IV, V, VI, VII,VIII, or IX (0.03-10 mg/kg) s.c. or p.o. and a control group is givensaline for 2-8 weeks. At 2, 4, 6 and 8 weeks animals are tested in therotating pole or cylinder test

Rotating pole: This test allows for the rapid assessment of coordinationand integration of motor movement, by the ability of the rat to traversea rotating pole as described previously (Johansson and Ohlsson, (1995)Stroke 26: 644-649. The pole has a length of 1500 mm, is elevated 750 mmabove the floor and rotates at 10 rpm to the right or left,respectively.

A score of 6-0 is given for each direction:

-   -   6, the animal crosses the pole with no foot slips;    -   5, the animal crosses the pole with a few foot slips;    -   4, the animal crosses the pole, slipping 50% of the footsteps;    -   3, the animal crosses the pole with more than 50% foot slips;    -   2, the animal walks a bit and then rotates around the pole;    -   1, the animal rotates around the pole without crossing it;    -   0, the animal falls off the pole.        Cylinder Test for Asymmetric Use of Forelimb Use in Spontaneous        Rearing

The cylinder test (modified from Schallert and Tillerson (Innovativemodels of CNS disease: from molecule to therapy. Clifton, N.J., Humana,1999) is used to quantify the forelimb use for rearing on the cylinderwall. The rats are monitored as they move freely in a 20-cm-wide clearglass cylinder. Contacts made by each forepaw with the cylinder wallwhile rearing are scored by a blinded observer. A total of 20 contactsare recorded for each animal, and the number of impaired (left), both,and non-impaired forelimb contacts as percentage of total contacts iscalculated. Baseline for rats is achieved by measuring the contacts madeby each forepaw before MCAO.

When using the rotating pole test or the cylinder test, the group ofanimals that are given compounds I, II, III, IV, V, VI, VII, VIII, or IXperform better than the group 1 control animals. Thus, animals fromcentral nervous system disorders show enhanced functional recovery whenadministered sigma receptor ligands.

Example 2

Evidence of Neuronal Regeneration in Rats Treated with Siramesine

1. Morris Water Maze

The Morris water maze was used to assess functional recovery, inparticular recovery in a cognitive skill, in hypertensive rats afterexposure to permanent middle artery occlusion (MCAO), a model ofischemic stroke.

Forty three hypertensive rats were exposed to permanent middle arteryocclusion (MCAO). Starting at two days after occlusion and continuingfor 14 days, Siramesine was administered p.o. in doses of 0.3 mg/kg (14rats) or 1.0 mg/kg (14 rats). In a control group (15 rats), vehicle onlywas administered. On day 35 after permanent MCAO, the rats were assessedfor their performance in the Morris Water Maze Test.

In the test, the rats were given a series of 6 trials, 1 hour apart in alarge dark-colored tank (200 cm in ) filled with clear water at atemperature of 22.0±1.5° C. A 12×12-cm submerged platform (2 mm belowthe water surface) was placed in the northwest quadrant of the pool. Therelease point was always the southern end of the pool. The rats werelowered into the pool facing the wall, and then released. Each rat wasgiven a maximum of 90 seconds to find the submerged platform. If it didnot find the platform in that time, the rat was physically guided to it.After remaining on the platform for 20 seconds, the rat was removed andplaced in a dry cage. One hour later, each rat was given a second trial,using the same release position and platform position, to measure therat's retention of the platform location. The process was repeated atotal of 5 times for each rat, each trial 1 hour apart. The swim pathsof the rats were recorded with a computer tracking system and thelatency and the length of swim path to find the hidden platform wereanalyzed. The latency to find the hidden platform (in seconds) is givenin Table 1. The length of swim path to find the hidden platform is givenin Table 2. TABLE 1 Latency to Find Hidden Platform LATENCY in SECONDS(SEM) DOSE TRIAL 1 TRIAL 2 TRIAL 3 TRIAL 4 TRIAL 5 VEHICLE 85.2 (3.6)69.3 (8.4) 63.4 44.69 52.7 (8.9) (8.8) (8.9) 0.3 mg/kg 72.4 (7.3) 43.6(7.9) 47.0 33.1 15.66 (8.7) (7.95) (2.2) 1.0 mg/kg 62.4 (7.7) 62.2 (8.5)59.99 34.3 19.8 (7.7) (6.7) (5.6)The treated animals performed better in all trials. The difference inlatency reached significance at trial 5 for both treatment groups and attrial 1 for the 1.0 mg/kg group.

Thus, this test that the sigma-2 selective agonist, siramesinefacilitates functional recovery, in particular recovery in a cognitiveskill, when administered daily to rats in a model of ischemic strokefrom 2 days after the stroke for 14 days. TABLE 2 Path Length to FindHidden Platform PATH LENGTH in METERS (SEM) DOSE TRIAL 1 TRIAL 2 TRIAL 3TRIAL 4 TRIAL 5 VEHICLE 15.7 (1.2) 19.0 (2.3)  18.7 (2.7) 13.8 (2.7)16.4 (3.0) 0.3 mg/kg 14.5 (1.8) 12.4 (2.3)  14.4 (2.7) 10.4 (2.4)  4.3(0.6) 1.0 mg/kg 11.1 (1.4) 16.2 (2.2) 16.65 (2.2)  9.7 (1.9) 5.29 (1.5)The treated animals performed better in all trials.

Example 3

Gene Expression Studies

The Ethics Committee for Animal Research at Lund University approved theexperimental protocol. Six-month-old SHR (spontaneous hypertensiverats), obtained from Mollegard Breeding Center, Ejby, Denmark, 2 monthsearlier and preoperatively housed in standard cages (550×350×200 mm, 3to 4 rats in each cage), were anesthetized with methohexital sodium(Brietal, 37° C.) 50 mg/kg intraperitoneally. The right MCA was accessedvia a small craniotomy, and the artery was ligated distal to thestriatal arteries, causing a neocortical infarct. The mean surgery timewas about 20 minutes and body temperature was maintained close to 37° C.Postoperatively, rats were kept in individual cages for 24 hours. Therats subjected to MCA occlusion (MCAO) were either returned to standardenvironment (SE), or were placed in a large, vertical,enriched-environment (EE) cage (815×610×1,280 mm), equipped withhorizontal and vertical boards, chains, swings, wooden blocks, andobjects of different sizes and materials. The distance between theboards and the movable objects was changed twice a week. The sham groupwere subjected to a sham surgery without MCAO and placed in the standardenvironment. In all experimental groups 12 and 60 days of recovery wereselected as end point analysis of gene expression. The study wasconducted using 6 experimental groups, with each group composed of 6-8animals.

The animals were sacrificed after 12 and 60 days from each experimentalgroup and tissues from the medial, rostral frontal cortex as well ashippocampus and striatum regions of the brain were isolated for RNApurification and target preparation. cDNA arrays consisting of 50,000clones from a rat cortex cDNA library were hybridized with labeledtarget nucleic acid obtained from control, standard MCAO and enrichedenvironment animals. About 3400 upregulated genes were selected afterbioinformatics analysis of the resulting gene discovery array data. Theraw clone data was normalized by the median empty well value for eachrespective array filter. These values were then transformed (log2+1) toapproximate normality. All replicates where then pooled for subsequentstatistical analysis. For each brain region (frontal, medial, rostral,hippocampus and striatum) the time points of the three experimentalconditions (enriched environment, stroke and control) where analyzed byprincipal component analysis (PCA). The outlying data points whereremoved from the data set. Next an ANOVA was performed with regard to aclone's behavior among the experimental conditions within a given brainregion and time point. The results of the ANOVA were filtered for clonesthat had a p-value less than 0.05. This filtered ANOVA list was thenanalyzed with the Tukey HSD test to determine the clone's expressionpattern.

Selection was based expression upregulation of±1.8-fold and acoefficient of variation (cv) of<0.2. The selected clones were picked,amplified by PCR, and re-printed on nylon membranes for profilingarrays. The profiling arrays were probed with probes from differentcortical and subcortical regions and recovery times of 12 and 60 days.Clones that were upregulated were selected for further analysis.

Analysis of the data discovery and profiling arrays allowed for theidentification of potential mechanistic pathways in the pathophysiologyof ischemic stroke and intracellular mechanism of functional recoveryafter enriched-environment. This analysis included principal componentanalysis of regulated clones, as well as clustering of regulated geneswith similar expression profiles. Principal component analysis yields acausality relationship among sets of genes clusters. Selection ofregulated genes as potential intervention targets for Central NervousSystem (CNS) disorders included, a series of criteria including, but notlimited to, sequence annotation, expression profile, placement of thegene within mechanistic pathways of biological relevance in thepathology of CNS disorders, technical feasibility to develop drugsdirected to modulate the specific gene (i.e. drugability), knownbiological role of a gene in CNS or other organ pathologies.

Analysis of EE array data demonstrated that in striatum and frontalcortex, type 1 sigma receptor mRNA is while in medial cortex, type 1sigma receptor mRNA is downregulated when the animals were placed in anenriched compared to standard environment. Thus, stimulation of brain byapplication of an enriched environment induces expression of the type 1sigma receptor in brain regions important for control of sensory-motorfunctions.

Example 4

Preparation of Tablets

The compound of formula V (10.0 g) is mixed with lactose (85.5 g),hydroxypropyl cellulose HPC-SL (2.0 g), hydroxypropyl cellulose L-HPC,LH-22 (2.0 g) and purified water (9.0 g), the resulting mixture issubjected to granulation, drying and grading, and the thus obtainedgranules are mixed with magnesium stearate (0.5 g) and subjected totablet making, thereby obtaining tablets containing 10 mg per tablet ofthe compound of formula V.

Example 5

Administering to a Subject

The tablet prepared in Example 4 is provided to a subject at time 0. Onetablet every 24 h is provided for a period of one week. Afteradministration of the third tablet, the subject is exposed to aneurodegenerative event. The treated subject exhibits symptoms ofneurological disorder that are less severe compared to the subject thatwas not treated.

All printed patents and referred to in this application are herebyincorporated herein in their entirety by this reference.

While the preferred of the invention has been illustrated and described,it will be appreciated that various changes can be made therein withoutdeparting from the spirit and scope of the invention.

1. A method of treating a mammalian subject to facilitate neuronalregeneration after onset of a neurodegenerative disease, which comprisesadministering to the subject an effective amount of siramesine, or saltsor solvates thereof.
 2. The method as claimed in claim 1, which furthercomprises assessing the subject for evidence of neuronal regeneration.3. The method as claimed in claim 2, wherein the evidence of neuronalregeneration is functional recovery in the subject.
 4. The method asclaimed in claim 3, wherein the functional recovery is recovery in amotor skill, cognitive skill, speech, sensory perception or sensoryfunction.
 5. The method as claimed in claim 4, wherein the functionalrecovery is recovery in a motor skill.
 6. The method as claimed in claim4, wherein the functional recovery is recovery in a cognitive skill. 7.The method as claimed in claim 2, wherein the evidence of neuronalregeneration is a structural change in the brain or spinal cord of thesubject.
 8. The method as claimed in claim 2, wherein siramesine isadministered repeatedly to the subject until evidence of neuronalregeneration is found.
 9. The method as claimed in claim 8, whereinsiramesine is administered daily.
 10. The method as claimed in claim 1,wherein the neurodegenerative disease is selected from ischemic stroke,traumatic brain injury and spinal cord injury.
 11. The method as claimedin claim 10, wherein the treatment is started at least 24 hours afterthe ischemic stroke, traumatic brain injury or spinal cord injury. 12.The method as claimed in claim 10, wherein the treatment is started atleast 48 hours after the ischemic stroke, traumatic brain injury orspinal cord injury.
 13. The method as claimed in claim 1, which furthercomprises exposing the subject to a stimulating environment.
 14. Themethod as claimed in claim 1, wherein the mammalian subject is a human.15. A pharmaceutical composition, which comprises siramesine, or saltsor solvates thereof, for treating a mammalian subject to facilitateneuronal regeneration after onset of a neurodegenerative disease, and apharmaceutically acceptable excipient.
 16. The pharmaceuticalcomposition as claimed in claim 15, which further comprises assessingthe subject for evidence of neuronal regeneration.
 17. Thepharmaceutical composition as claimed in claim 16, wherein the neuronalregeneration is evidenced by functional recovery.
 18. The pharmaceuticalcomposition as claimed in claim 17, wherein the functional recovery isrecovery in a motor skill, cognitive skill, speech, sensory perceptionor sensory function.
 19. The pharmaceutical composition as claimed inclaim 18, wherein the functional recovery is recovery in a motor skill.20. The pharmaceutical composition as claimed in claim 18, wherein thefunctional recovery is recovery in a cognitive skill.
 21. Thepharmaceutical composition as claimed in claim 16, wherein the neuronalregeneration is evidenced by a structural change in the brain or spinalcord of the subject.
 22. The pharmaceutical composition as claimed inclaim 15, wherein siramesine is administered repeatedly to the subjectuntil evidence of neuronal regeneration is found.
 23. The pharmaceuticalcomposition as claimed in claim 22, wherein siramesine is administereddaily.
 24. The pharmaceutical composition as claimed in claim 15,wherein the neurodegenerative disease is selected from ischemic stroke,traumatic brain injury and spinal cord injury.
 25. The pharmaceuticalcomposition as claimed in claim 24, wherein the treatment is started atleast 24 hours after the ischemic stroke, traumatic brain injury orspinal cord injury.
 26. The pharmaceutical composition as claimed inclaim 24, wherein the treatment is started at least 48 hours after theischemic stroke, traumatic brain injury or spinal cord injury.
 27. Thepharmaceutical composition as claimed in claim 15, which furthercomprises exposing the subject to a stimulating environment.
 28. Thepharmaceutical composition as claimed in claim 15, wherein the mammaliansubject is a human.
 29. A method for facilitating functional recoveryafter onset of a neurodegenerative disease in a mammalian subject inneed thereof, the method comprising: administering a pharmaceuticallyeffective amount of siramesine, or salts or solvates thereof, to thesubject.
 30. The method of claim 29, which further comprises exposingthe subject to a stimulating environment.
 31. The method of claim 30,wherein the neurodegenerative disease is selected from the groupconsisting of ischemic stroke, traumatic brain injury and spinal cordinjury.
 32. The method of claim 31, wherein the neurodegenerativedisease is ischemic stroke.
 33. The method of claim 31, wherein theligand is administered during or immediately after a stroke.
 34. Themethod of claim 31, wherein siramesine is administered within 24 hoursof the disease onset.
 35. The method of claim 31, wherein siranesine isadministered within 48 hours of the disease onset.
 36. The method ofclaim 31, wherein siramesine is administered for at least 2 weeks. 37.The method of claim 31, wherein siramesine is administered for 1 month.38. The method of claim 31, wherein siramesine is administered for 3months.
 39. The method of claim 31, wherein siramesine is administeredfor 3 months or longer.
 40. The method of claim 31, wherein siramesineis administered continuously.
 41. The method of claim 31, wherein thesubject is human.